The Role and Regulation of TRP53 Activity in Oocytes and Granulosa Cells After Radiation-induced Damage

项目摘要 电离辐射(IR)是一种用于抗癌和在治疗前切除患者骨髓的治疗方法 造血干细胞移植。红外线导致损伤(双链断裂或活性氧物种) 但也可以杀死健康的旁观者细胞。原始卵泡(PFS)是一种有限的卵巢 含有未成熟卵母细胞且极易受损的卵泡。红外线或其他因素造成的损坏 基因毒性化疗可以耗尽PFS的卵巢，导致卵巢早衰(POI)和 不孕不育。因此，有必要了解损伤是如何影响卵巢细胞并导致POI的 可以开发毒性较低的治疗性或预测性治疗方法。该项目的目标是确定 辐射诱导哺乳动物卵母细胞消除的机制。PF对损害的反应在很大程度上是 除了几个主要玩家之外，其他人都不知道。Checkpoint kinase2(CHEK2)主要激活促凋亡的TRP63 TA异构体(TAp63)对损伤的反应。相比之下，体细胞主要使用另一个CHEK2靶标 TrP53。我们的实验室显示，接受IR的CHEK2-/-雌性小鼠保留了它们的PF储备，并产生了健康的幼鼠。 接受低剂量IR的雌性TAp63-/-维持了PFS，而TrP53-/-小鼠失去了PF储备。暗示着 Trp53在卵母细胞的凋亡过程中起着不可或缺的作用。在IR剂量较高的治疗或化疗中，TAp63-/- 雌性失去Pf储备，而CHEK2-/-雌性保留Pf储备。我们预测了更高剂量的IR和 化疗会造成更大的损害，从而激活TAp63不依赖的机制。的确，TAp63-/- 暴露于较高剂量IR的Trp53-/-双突变雌性保留了PF储备，提示Trp53触发了 当达到一定的损害阈值时，卵母细胞就会被清除。术后TrP53蛋白表达的分析 高剂量红外光谱分析表明，在纯化的卵母细胞中存在一种独特的TrP53蛋白，并且没有典型的~53 kDa蛋白。 在体细胞中检测到。基于这些观察，我们假设卵母细胞中的Trp53活性是受调控的 通过卵母细胞特有的机制激活Trp53或将其作用限制到特定的阈值 损害就会发生。这项提议将利用遗传方法(嵌合重组卵巢)和蛋白质组学。 方法(质谱学)，以确定TrP53的调节对卵巢损伤的反应。的目标是 这项拟议的研究旨在(1)确定Trp53依赖的PF丢失是否在 卵母细胞或由于体细胞颗粒细胞对卵母细胞的损伤信号以及2)Trp53如何决定 通过识别独特的翻译后修饰和/或蛋白质相互作用来调节卵母细胞的活性 与TrP53依赖的PF缺失相关。确定卵母细胞特有的促凋亡调控机制 TrP53对损伤的反应将提高我们对Pf对基因毒性药物的反应的理解 并为治疗提供靶点，以防止接受基因毒性治疗的患者的卵母细胞丢失、不孕和POI 治疗。

Project Summary Ionizing radiation (IR) is a treatment used against cancer and to ablate patient's bone marrow prior to hematopoietic stem cell transplantation. IR induces damage (double strand breaks or reactive oxygen species) in target cells but can also kill healthy bystander cells. Primordial follicles (PFs) are a limited population of ovarian follicles that contain immature oocytes and are highly susceptible to damage. Damage induced by IR or other genotoxic chemotherapies can deplete the ovary of PFs resulting in premature ovarian insufficiency (POI) and infertility. Therefore, there is a need to understand how damage affects ovarian cells and leads to POI before less toxic therapeutic or predictive treatments can be developed. The goal of this project is to determine the mechanism of radiation-induced oocyte elimination in mammals. How PF's response to damage is largely unknown beyond a few major players. Checkpoint kinase 2 (CHEK2) primarily activates the pro-apoptotic TRP63 TA isoform (TAp63) in response to damage. In contrast, somatic cells predominantly use another CHEK2 target TRP53. Our lab showed Chek2-/- female mice receiving IR retained their PF reserve and produced healthy pups. TAp63-/- females receiving low dose IR maintained PFs while Trp53-/- mice lost their PF reserve. Suggesting that TRP53 is dispensable for apoptosis in oocytes. In treatments with higher IR dose or chemotherapies, TAp63-/- females lose PF reserve while Chek2-/- females retain PF reserve. We predicted higher doses of IR and chemotherapies cause more damage, which activates a TAp63-independent mechanism. Indeed, TAp63-/- Trp53-/- double mutant females exposed to higher dose IR retained PF reserve suggesting TRP53 triggers oocyte elimination when a certain threshold of damage is reached. Analysis of TRP53 protein expression after high dose IR identified a unique form of TRP53 in purified oocytes and the absence of the typical ~53kDa protein, detected in somatic cells. Based on these observations we hypothesize TRP53 activity in oocytes is regulated by an oocyte-specific mechanism which either activates TRP53 or restricts its action until a specific threshold of damage occurs. This proposal will utilize genetic approaches (chimeric reconstituted ovaries) and proteomic approaches (mass spectrometry) to determine TRP53 regulation in response to ovarian damage. The aims of this proposed research are to (1) determine whether TRP53-dependent PF loss is triggered intrinsically in the oocyte or is due to damage signals from somatic granulosa cells to the oocyte and 2) determine how TRP53 activity is regulated in oocytes by identifying unique post-translational modifications and/or protein interactions associated with TRP53-dependent PF loss. Defining oocyte-specific mechanisms regulating pro-apoptotic TRP53 activity in response to damage will improve our understanding of the PF response to genotoxic agents and provide targets for therapeutics to prevent oocyte loss, infertility, and POI in patients receiving genotoxic treatments.